Fig 6.
The LP/SP reporter system demonstrates differences in the behavior of the 5′ UTRs. (A) Schematic representation of the SPACER 50 insertion in the context of 5′ UTRL-LPNext (L) and 5′ UTRS-LPNext (S). (B) The SPACER 0 (WT, represented by L and S) and SPACER 50 (L+50 and S+50) constructs were expressed in HEK293T cells. Proteins were visualized by immunoblotting with an anti-HA antibody. Lane Δ, a cell extract prepared from ΔKpn-LPNext-transfected cells that provides a marker for protein products arising from all the AUG codons depicted in panel A. Transfections were performed in duplicate. The protein bands in each individual transfection arising from the AUGELK-1 and iAUGa/b/c were quantified. The value of their ratio in each transfection is indicated below the gel (averaged from the duplicate samples). (C) Schematic representation of the impact of the uORF2 UGA/C mutation in the 5′ UTRL-LPNext and 5′ UTRS-LPNext reporter system. The sequence of the uORF2 is indicated, as is the distance between the stop codon of this ORF and the downstream AUG codons (arrows). (D) HEK293T cells were transfected with ΔKpn-LPNext (lanes Δ), 5′ UTRL-LPNext (lanes L), 5′ UTRS-LPNext (lanes S), and the long and short 5′ UTRs carrying the UGA/C mutation (lanes L-UGA/C and S-UGA/C). Protein products were detected by immunoblotting with the anti-HA antibody. Lane M, the same marker for LPNext and LP as outlined in the legend to Fig. 5. The AUGELK-1/iAUGa/b/c levels within each duplicate transfection were determined, and the averaged value is indicated below the gel. (Lower inset) An overexposure of the L-UGA/C and S-UGA/C lanes demonstrating the presence of a protein product arising from the AUGELK-1.